The present invention relates to novel receptors in the TNF family. A novel receptor has been identified, referred to herein as TRAIN (TNF Receptor in the BRAIN).
The TNF family consists of pairs of ligands and their specific receptors referred to as TNF family ligands and TNF family receptors (Bazzoni and Beutler, 1996. N Engl J Med 334, 1717-25). The family is involved in the regulation of the immune system and possibly other non-immunological systems. The regulation is often at a “master switch” level such that TNF family signaling can result in a large number of subsequent events best typified by TNF. TNF can initiate the general protective inflammatory response of an organism to foreign invasion that involves the altered display of adhesion molecules involved in cell trafficking, chemokine production to drive specific cells into specific compartments and the priming of various effector cells. As such, the regulation of these pathways has clinical potential.
The TNF receptor family is a collection of related proteins that generally consist of an extracellular domain, a transmembrane domain and an intracellular signaling domain. The extracellular domain is built from 2-6 copies of a tightly disulphide bonded domain and is recognized on the basis of the unique arrangement of cysteine residues. Each receptor binds to a corresponding ligand although one ligand may share several receptors. In some cases, it is clear that by alternate RNA splicing, soluble forms of the receptors lacking the transmembrane region and intracellular domain exist naturally. Moreover, in nature, truncated versions of these receptors exist and the soluble inhibitory form may have direct biological regulatory roles. Clearly, viruses have used this tactic to inhibit TNF activity in their host organisms (Smith, 1994. Trends in Microbiol. 82, 81-88). These receptors can signal a number of events including cell differentiation, cell death or cell survival signals. Cell death signaling often is triggered via relatively direct links to the caspase cascade of proteases e.g. Fas and TNF receptors. Most receptors in this class can also activate NFKB controlled events.
An emerging theme in the TNF family of receptors has been the use by nature of both full length receptors with intracellular domains that transmit a signal and alternate forms which are either secreted or lack an intracellular signaling domain. These later forms can inhibit ligand signaling and hence can dampen a biological response. There are several examples of this phenomenon. First, the TNF receptor p75 is readily secreted following selective cleavage from the membrane and then acts to block the action of TNF. It is likely that nature has evolved this system to buffer TNF activity. A second example is provided by the TRIAL-TRAIL receptor system where there are 4 separate genes encoding TRAIL receptors. Two of these TRAIL-R1 and TRAIL-R2 possess intracellular domains and transduce signal. A third receptor (TRAIL-R4) has an intracellular domain yet this domain does not have all the elements is found in R1 and R2, e.g. it lacks a domain capable of signaling cell death. Lastly, there is a fourth receptor TRAIL-R3, that is essentially a soluble form but remains tethered by a glycolipid linkage. Hence this receptor can bind ligand yet it is unable to transmit a signal, i.e. it is effectively a decoy receptor. A third example is provided by the osteoprotegerin (OPG) system where the OPG receptor lacks a transmembrane domain and is secreted into the medium. This receptor can block the signaling necessary to induce osteoclast differentiation possibly by binding to a ligand called RANK-L. The TRAIN system described here resembles the OPG paradigm in that a short version can be secreted that would inhibit the natural TRAIN-L (currently unknown) from binding to full length TRAIN and eliciting a signal.
The receptors are powerful tools to elucidate biological pathways via their easy conversion to immunoglobulin fusion proteins. These dimeric soluble receptor forms are good inhibitors of events mediated by either secreted or surface bound ligands. By binding to these ligands they prevent the ligand from interacting with cell associated receptors that can signal. Not only are these receptor-Ig fusion proteins useful in an experimental sense, but they have been successfully used clinically in the case of TNF-R-Ig to treat inflammatory bowel disease, rheumatoid arthritis and the acute clinical syndrome accompanying OKT3 administration (Eason et al., 1996. Transplantation 61, 224-8; Feldmann et al., 1996. Annu Rev Immunol; van Dullemen et al., 1995. Gastroenterology 109, 129-35). One can envision that manipulation of the many events mediated by signaling through the TNF family of receptors will have wide application in the treatment of immune based diseases and also the wide range of s human diseases that have pathological sequelae due to immune system involvement. A soluble form of a recently described receptor, osteoprotegerin, can block the loss of bone mass and, therefore, the events controlled by TNF family receptor signaling are not necessarily limited to immune system regulation. Antibodies to the receptor can block ligand binding and hence can also have clinical application. Such antibodies are often very long-lived and may have advantages over soluble receptor-Ig fusion proteins which have shorter blood half-lives.
While inhibition of the receptor mediated pathway represents the most exploited therapeutic application of these receptors, originally it was the activation of the TNF receptors that showed clinical promise (Aggarwal and Natarajan, 1996. Eur Cytokine Netw 7, 93-124). Activation of the TNF receptors can initiate cell death in the target cell and hence the application to tumors was and still is attractive (Eggermont et al., 1996. J Clin Oncol 14, 2653-65). The receptor can be activated either by administration of the ligand, i.e. the natural pathway or some antibodies that can crosslink the receptor are also potent agonists. Antibodies would have an advantage in oncology since they can persist in the blood for long periods whereas the ligands generally have short lifespans in the blood. As many of these receptors may be expressed more selectively in tumors or they may only signal cell death or differentiation in tumors, agonist antibodies could be good weapons in the treatment of cancer. Likewise, many positive immunological events are mediated via the TNF family receptors, e.g. host inflammatory reactions, antibody production etc. and therefore agonistic antibodies could have beneficial effects in other, non-oncological applications.
Paradoxically, the inhibition of a pathway may have clinical benefit in the treatment of tumors. For example the Fas ligand is expressed by some tumors and this expression can lead to the death of Fas positive lymphocytes thus facilitating the ability of the tumor to evade the immune system. In this case, inhibition of the Fas system could then allow the immune system to react to the tumor in other ways now that access is possible (Green and Ware, 1997. Natl. Acad. Sci. USA 94, 5986-5990).
The receptors are also useful to discover the corresponding ligand as they can serve as probes of the ligand in expression cloning techniques (Smith et al., 1993. Cell 73, 1349-60). Likewise, the receptors and ligands can form in vitro binding assays that will allow the identification of inhibitory substances. Such substances can form the basis of novel inhibitors of the pathways.